Gene therapy of tumors with an endothelial cell-specific, cell cycle-dependent active compound

ABSTRACT

A DNA sequence for the gene therapy of tumors is described. In its essential elements, the DNA sequence is composed of an activator sequence, a promoter module and a gene for the active substance. The activator sequence is activated, in a cell-specific manner, in proliferating endothelial cells or in cells which are adjacent to these endothelial cells. This activation is regulated by the promoter module in a cell cycle-specific manner. The active substance is an inhibitor of angiogenesis or a cytostatic or cytotoxic molecule. The DNA sequence is inserted into a viral or non-viral vector which is supplemented with a ligand which possesses affinity for the activated endothelial cell.

TECHNICAL FIELD

A DNA sequence for the gene therapy of tumors is described. In itsessential elements, the DNA sequence is composed of an activatorsequence, a promoter module and a gene for the active substance.

The activator sequence is activated, in a cell-specific manner, inproliferating endothelial cells or cells which are adjacent to theseendothelial cells.

This activation is regulated in a cell cycle-specific manner by thepromoter module.

The active substance is an inhibitor of angiogenesis or a cytostatic orcytotoxic molecule. The DNA sequence is inserted into a viral ornon-viral vector which is supplemented with a ligand which has affinityfor the activated endothelial cell.

1.Tumor Growth and Angiogenesis

The deficient activity of many antitumoral active compounds can beexplained, at least to some degree, by the fact that the tumor cellswithin a tumor node are inaccessible to the antitumoral, in particularhigh molecular weight, active compounds (Burrows et al., Pharm. Ther.64, 155 (1994), Baxter et al., Microvasc. Rex. 41, 5 (1991)). Suchactive compounds have to diffuse through the vascular endothelium andthe basal membrane, and through the tumor stroma and tumor parenchyma,in order to reach each individual tumor cell. The extent of thisdiffusion is essentially determined by the concentration or theconcentration gradient of the active compound and its physicochemicalcharacteristics. Moreover, convection, which is directed outwards by thehigher pressure in the interior of the tumor (Burrows et al., Microvasc.Res. 41, 5 (1991)), runs counter to the diffusion.

Since tumor blood vessels are accessible even to high molecular weightactive compounds, it was consequently proposed at an early stage(Denekamp, Prog. Appl. Microcirc. 4, 28 1984), Denekamp, Cancer Topics6, 6 1986), Denekamp, Cancer Metast. Rev. 9, 267 (1990), Denekamp, Brit.J. Radiol. 66, 181 (1993)), that use be made of the angiogenesis whichis induced by tumors for tumor therapy.

Thus, attempts were made to inhibit tumor growth using substances whichinhibit angiogenesis (Bicknell et al., Semin. Cancer Biol. 3, 399(1992)). Experimental investigations in animals demonstrated thatsystemic administration of substances which inhibit angiogenesis canalso inhibit tumor proliferation. This applies, for example, to suramin(Gagliardi et al., Cancer Rex. 52, 5073 (1992)), to heparin/steroidconjugates (Thorpe et al., Cancer Res. 53, 3000 (1993)), toO-(chloroacetylcarbamoyl)fumigillol (Yamaoka et al., Cancer Res. 53,4262 (1993)), to monoclonal antibodies against angiogenin (Olson et al.,Cancer Res. 54, 4576 (1994)) and to angiostatin (O'Reilly et al., Cell79, 315 (1994)).

However, the abovementioned methods suffered from the disadvantages ofthe systemic, non-tumor-specific effect of the angiogenesis inhibitors,their side effects and the risk of fresh tumor growth occurring once thetherapy was discontinued.

As an alternative, the idea was conceived of inhibiting the blood supplyof the tumors by damaging endothelial cells so that the tumors necrose(Denekamp, Brit. J. Radiol. 66, 181 (1993)). With this idea in mind, theadministration was proposed of toxins, cytostatic agents or isotopeswhich were coupled to antibodies. These antibodies would be specific forthe tumor-associated vascular endothelium. The intention was that theantibody conjugates would destroy the tumor-associated blood vessel inan endothelium-specific manner and thereby induce necrosis of the tumor(Burrows et al., Pharma Ther. 64, 155 (1994)).

A further suggestion was to bind thrombogenic substances, cytokines orchemokines to tumor-associated endothelial cells by way of specificantibodies and to exert an influence on tumor growth by way of the bloodcoagulation, inflammation or immunoregulation which was elicitedthereby. Similar effects were sought by the proposal to introduce DNAinto endothelial cells by way of endothelial cell-specific antibodieswith the DNA transducing the endothelial cells to secrete inflammatoryor immuno-regulatory substances or substances which affected the growthof tumor cells (Burrows et al., Pharmac. Ther. 64, 155 (1994)).

Membrane antigens on the surface of endothelial cells were proposed asantigens for antibodies of this nature. These antigens include, forexample, endoglin, endosialin, p96 dimer, VEGF receptors, PDGFreceptors, urokinase (uPA) receptors and various adhesion molecules(Burrows et al., Pharmac. Ther. 64, 155 (1994)).

However, a feature possessed by all these membrane antigens is that theyare also present on non-proliferating endothelial cells, at least inrelatively low concentrations. Since non-proliferating endothelial cellsfar outnumber proliferating endothelial cells even in a tumor-affectedorganism, this does not then adequately ensure the requisite tumorspecificity of the effect which is sought by administering the antibodyconjugate.

2. Description of the Active Compound

The present invention now relates to an active compound (i.e. apharmaceutical) which can be given to tumor patients both locally andsystemically and which results, at the site of the tumor growth,predominantly if not exclusively in the proliferating endothelial cellsand over a relatively long period of time, in angiogenesis beinginhibited, in an inflammation being induced or in cytostatic substancesbeing generated directly or indirectly, thereby inhibiting growth of thetumor.

The central component of this active compound is a DNA construct whichis composed of the following elements: ##STR1## (Throughout the text ofthis application, DNA is used as a common term both for a complementary(cDNA) and a genomic DNA sequence).

2.1 Choice of the Activator Sequence

An activator sequence is to be understood to be a nucleotide sequence(promoter sequence or enhancer sequence) with which transcriptionfactors interact which are formed or are active either in endothelialcells or else in cells which are in the immediate vicinity ofproliferating endothelial cells.

a) Activator sequences which are activated in endothelial cells

The CMV enhancer, the CMV promoter (EP 0173.177.B1), the SV40 promoter,or any other promoter sequence or enhancer sequence which is known tothe skilled person, may be used as the activator sequence.

However, within the meaning of this invention, the preferred activatorsequences include those gene-regulatory sequences or elements from geneswhich encode proteins which can be detected, in particular, inendothelial cells (or else in cells in the immediate vicinity ofproliferating endothelial cells).

Some of these proteins have been described by Burrows et al. (Pharmac.Therp. 64, 155 (1994)) and Plate et al. (Brain Pathol. 4, 207 (1994)).Particular examples of these endothelial cell-specific proteins are:

brain-specific, endothelial glucose-1-transporter

Endothelial cells of the brain are notable for very strong expression ofthis transporter for the purpose of effecting transendothelial transportof D-glucose into the brain (Gerhart et al., J. Neurosci. Res. 22, 464(1989)). The promoter sequence has been described by Murakami et al. (J.Biol. Chem. 267, 9300 (1992)).

endoglin

Endoglin appears to be a non-signal transmitting receptor of TGFβ(Gougos et al., J. Biol. Chem. 265, 8361 (1990), Cheifetz, J. Biol.Chem. 267, 19027 (1992), Moren et al., BBRC 189, 356 (1992)). While itis present in low quantities on normal endothelium, it is expressed toan increased extent on proliferating endothelium (Westphal et al., J.Invest. Derm. 100, 27 (1993), Burrows et al., Pharmac. Ther. 64, 155(1994)). The promoter sequence has been described by Bellon et al. (Eur.J. Immunol. 23, 2340 (1993)) and Ge et al. (Gene 138, 201 (1994)).

VEGF receptors

Two receptors are distinguished (Plate et al., Int. J. Cancer 59, 520(1994)):

VEGF receptor 1 (flt-1) (de Vries et al., Science 255, 989 (1992)(contains an fms-like tyrosine kinase in the cytoplasmic moiety) and the

VEGF receptor 2 (flk-1, KDR) (Terman et al., BBRC 187, 1579 (1992))(contains a tyrosine kinase in the cytoplasmic moiety).

Both receptors are to be found almost exclusively on endothelial cells(Senger et al., Cancer Metast. Rev. 12, 303 (1993)).

other endothelium-specific receptor tyrosine kinases

til-1 or til-2 (Partanen et al., Mol. Cell Biol. 12, 1698 (1992),Schnurch and Risau, Development 119, 957 (1993), Dumont et al., Oncogene7, 1471 (1992))

B61 receptor (Eck receptor) (Bartley et al., Nature 368, 558 (1994),Pandey et al., Science 268, 567 (1995), van der Geer et al., Ann. Rev.Cell Biol. 10, 251 (1994))

B61

The B61 molecule represents the ligand for the B61 receptor. (Holzman etal., J. Am. Soc. Nephrol. 4, 466 (1993), Bartley et al., Nature 368, 558(1994))

endothelin, especially

endothelin, B (Oreilly et al., J. Cardiovasc. Pharm. 22, 18 (1993),Benatti et al., J. Clin. Invest. 91, 1149 (1993), O'Reilly et al., BBRC193, 834 (1993). The promoter sequence has been described by Benatti etal., J. Clin. Invest. 91, 1149 (1993).

endothelin 1 (Yanasigawa et al., Nature 332, 411 (1988). The promotersequence has been described by Wilson et al., Mol. Cell. Biol. 10, 4654(1990).

endothelin receptors, in particular the endothelin B receptor (Webb etal., Mol. Pharmacol. 47, 730 (1995), Haendler et al., J. Cardiovasc.Pharm. 20, 1 (1992)).

mannose-6-phosphate receptors (Perales et al., Eur. J. Biochem. 226, 225(1994). The promoter sequences have been described by Ludwig et al.(Gene 142, 311 (1994)), Oshima et al. (J. Biol. Chem. 263, 2553 (1988))and Pohlmann et al. (PNAS USA 84, 5575 (1987)).

von Willebrand factor

The promoter sequence has been described by Jahroudi and Lynch (Mol.Cell. Biol. 14, 999 (1994), Ferreira et al., Biochem. J. 293, 641 (1993)and Aird et al., PNAS USA 92, 4567 (1995)).

IL-1α and IL-1β

IL-1 is produced by activated endothelial cells (Warner et al., J.Immunol. 139, 1911 (1987)).

The promoter sequences have been described by Hangen et al., Mol.Carcinog. 2, 68 (1986), Turner et al., J. Immunol. 143, 3556 (1989),Fenton et al., J. Immunol. 138, 3972 (1987), Bensi et al., Cell GrowthDiff. 1, 491 (1990), Hiscott et al., Mol. Cell. Biol. 13, 6231 (1993)and Mori et al., Blood 84, 1688 (1994).

IL-1 receptor The promoter sequence has been described by Ye et al.,PNAS USA 90, 2295 (1993).

vascular cell adhesion molecule (VCAM-1) The expression of VCAM-1 inendothelial cells is activated by lipopolysaccharides, TNF-α (Neish etal., Mol. Cell. Biol. 15, 2558 (1995)), IL-4 (lademarco et al., J. Clin.Invest. 95, 264 (1995)), IL-1 (Marni et al., J. Clin. Invest. 92, 1866(1993)). The promoter sequence of VCAM-1 has been described by Neish etal., Mol. Cell. Biol. 15, 2558 (1995), Ahmad et al., J. Biol. Chem. 270,8976 (1995), Neish et al., J. Exp. Med. 176, 1583 (1992), lademarco etal., J. Biol. Chem. 267, 16323 (1992) and Cybulsky et al., PNAS USA 88,7859 (1991).

Synthetic activator sequence As an alternative to naturalendothelium-specific promoters, synthetic activator sequences can alsobe used which are composed of oligomerized binding sites fortranscription factors which are preferentially or selectively active inendothelial cells. An example of such a transcription factor is thetranscription factor GATA-2, whose binding site in the endothelin 1 geneis 5'-TTATCT-3' (Lee et at., Biol. Chem. 266, 16188 (1991), Dorfmann etal., J. Biol. Chem. 267, 1279 (1992) and Wilson et al., Mol. Cell. Biol.10, 4854 (1990)).

b) Activator sequences which are activated in cells in the vicinity ofactivated endothelial cells

In proliferating endothelia, neighboring cells become accessible tomacromolecules of the blood through tight junctions which are opened. Asa result of the functional and anatomical interrelationships, theneighboring cells of activated endothelial cells are target cells withinthe meaning of this invention.

VEGF

VEGF is formed by various cells (e.g. smooth muscle cells and tumorcells) in the immediate vicinity of proliferating endothelial cells,particularly under hypoxic conditions (Ferrara et al., Endoc. Rev. 13,18 (1992), Berse et al., Mol. Biol. Cell 3, 211 (1992), Finkenzeller etal., BBRC 208, 432 (1995), Tischer et al., BBRC 165, 1198 (1989), Leunget al., Science 246, 1306 (1989)). The gene-regulatory sequences for theVEGF gene are

the promoter sequence of the VEGF gene (5'-flanking region) (Michenko etal., Cell Mol Biol. Res. 40, 35 (1994), Tischer et al., J. Biol. Chem.266, 11947 (1991)) or

the enhancer sequence of the VEGF gene (3'-flanking region) (Michenko etal., Cell Mol. Biol. Res. 40, 35 (1994) or

the c-Src gene (Mukhopadhyay et al., Nature 375, 577 (1995), Bonham etal., Oncogene 8, 1973), Parker et al., Mol. Cell. Biol. 5, 831 (1985),Anderson et al., Mol. Cell. Biol. 5, 1122 (1985)) or

the V-Src gene (Mukhodpadhyay et al., Nature 375, 577 (1995), Andersonet al., Mol. Cell. Biol. 5, 1122 (1985), Gibbs et al., J. Virol. 53, 19(1985))

Steroid hormone receptors and their promoter elements (Truss and Beato,Endocr. Rev. 14, 459 (1993)), in particular the

Mouse mammary tumor virus promoter In most cells, this promoter isactivated by steroids, for example by glucocorticosteroids (Parks etal., Cell 8, 87 (1976)) or by progestins (Cato et al., EMBO J. 5, 2237(1986)). The cDNA sequence of the promoter region of the long terminalrepeat region of MMTV has been described by Chalepakis et al., Cell 53,371 (1988) and Truss and Beato (Endocr. Rev. 14, 459 (1993).

2.2. Choice of the Repressor Module

As an example, a cell cycle-regulated promoter module is to beunderstood to be the nucleotide sequence CDE-CHR-lnr (see below). Theessential function of this promoter module is to inhibit the function ofthe activator sequence in the G0/G1 phase of the cell cycle and totrigger cell cycle-specific expression in the S/G2 phase andconsequently in proliferating cells.

The promoter module CDE-CHR-lnr was discovered in the context of adetailed investigation of the G2-specific expression of the human cdc25Cpromoter. The starting point was the discovery of a repressor element(cell cycle dependent element; CDE) which is responsible for switchingoff the promoter in the G1 phase of the cell cycle (Lucibello et al.,EMBO J. 14, 132 (1995)). By means of genomic dimethyl sulfate (DMS)footprinting and functional analyses (FIGS. 1 and 2), it was possible todemonstrate that CDE binds a repressor (CDE-binding factor; CDF) in aG1-specific manner and thereby gives rise to inhibition of transcriptionin non-proliferating (G0) cells. In its repressing function, the CDE,which is located in the region of the basal promoter, is dependent on anupstream activating sequence (UAS). This led to the conclusion that theCDE-binding factor inhibits the transcription-activating effect of5'-bound activator proteins in a cell cycle-dependent manner, i.e. innon-proliferating cells and in the G1 phase of the cell cycle (FIG. 3).

This conclusion was confirmed by a further experiment: Fusion of theviral, non-cell cycle-regulated early SV40 enhancer with a cdc25 minimalpromoter (composed of CDE and the 3'-situated start sites) led to clearcell cycle regulation of the chimeric promoter (FIG. 4). Subsequentinvestigations of the cdc25C enhancer have shown that the transcriptionfactors which are regulated by CDF in a cell cycle-dependent manner areNF-Y (synonym: CBF; Dorn et al., Cell 50, 863 (1987), van Hujisduijnenet al., EMBO J. 9, 3119 (1990), Coustry et al., J. Biol. Chem. 270, 468(1995)), Sp1 (Kadonaga et al., TIBS 11, 10, 1986)) and a factor (CIF)which is possibly novel and which binds to CBS7. An additionalinteresting finding of this study was the observation that NF-Y withinthe cdc25C enhancer only activates transcription efficiently incooperation with at least one further NF-Y complex or with CIF. BothNF-Y and Sp1 belong to the class of glutamine-rich activators, somethingwhich provides important information about the mechanism of repression(e.g. interaction or interference with particular basal transcriptionfactors or TAFs).

Comparison of the promoter sequences of cdc25C, cyclin A and cdc2 (SEQID NOS: 1-2, 5-6, and 3-4, respectively) demonstrated homologies inseveral regions (FIG. 5). Not only is the CDE conserved in all 3promoters (the divergences which are present are not functionallyrelevant) but the neighboring Y_(c) boxes are conserved as well. Asexpected, all these regions exhibited protein binding in vivo, with thisprotein binding being cell cycle-dependent in the case of the CDE. Inaddition, it was possible to demonstrate that all 3 promoters arederegulated by mutation of the CDE (Table 1). When the cdc25C, cyclin Aand cdc2 sequences were compared, a remarkable similarity was alsoevident in the region immediately 3' of the CDE (cell cycle geneshomology region; CHR) (FIG. 5). Although this region is functionally asimportant as the CDE (Table 1), it is not visible in the in-vivo DMSfootprinting experiments. A possible explanation for this is aninteraction of the factor with the minor groove of the DNA. Results fromelectrophoretic mobility shift assay (EMSA) experiments point to CDE andCHR together binding a protein complex, the CDF. These observationsindicate that CDF-mediated repression of glutamine-rich activators is afrequently occurring mechanism of cell cycle-regulated transcription.

However, it is apparently not only the CDE-CHR region which is ofimportance for regulating the cdc25C promoter but also one of theinitiation sites (position +1) within the nucleotide sequence of thebasal promoter (positions ≦-20 to ≧+30, see FIG. 1). Mutations in thisregion, which encompasses the in-vitro binding site for YY-1 (Seto andShenk, Nature 354, 241 (1991), Usheva and Shenk, Cell 76, 1115 (1994))lead to complete deregulation. In view of the proximity of the CDE-CHRto the basal promoter, interaction of the CDF with the basaltranscription complex is consequently very probable.

2.3. Choice of the Antitumoral Substance

a) Inhibitors of proliferation

Within the meaning of this invention, an antitumoral substance is to beunderstood as being the DNA sequence of a protein which inhibits theproliferation of endothelial cells. Examples of these DNA sequences arethe DNA sequences, which are listed in the following literaturereferences, for

retinoblastoma protein (pRb/p110) or for its analogs p107 and p120 (LaThangue, Curr. Opin. Cell Biol. 6, 443 (1994))

protein p53 (Prives et al., Genes Dev. 7, 529 (1993))

protein p21 (WAF-1) (El-Deiry et al., Cell 75, 817 (1993))

protein p16 (Serrano et al., Nature 366, 704 (1993), Kamb et al.,Science 264, 436 (1994), Nobori et al., Nature 368, 753 (1994))

other CdK inhibitors (review in Pines, TIBS 19, 143 (1995))

GADD45 protein (Papathanasiou et al., Mol. Cell. Biol. 11, 1009 (1991),Smith et al., Science 266, 1376 (1994))

bak protein (Farrow et al., Nature 374, 731 (1995) Chittenden et al.,Nature 374, 733 (1995), Kiefer et al., Nature 374, 736 (1995))

In order to prevent rapid intracellular inactivation of these cell cycleinhibitors, those genes should preferably be used which possessmutations for the inactivation sites of the expressed proteins withoutthe function of these proteins thereby being impaired.

Retinoblastoma protein (pRb) and the related p107 and p130 proteins areinactivated by phosphorylation. Consequently, a pRb/p110, p107 or p130CDNA sequence is used which is point-mutated such that thephosphorylation sites of the encoded protein are replaced with aminoacids which cannot be phosphorylated.

According to Hamel et al. (Mol. Cell. Biol. 12, 3431 (1992)),retinoblastoma protein (pRb/p110) can no longer be phosphorylated whenthe amino acids in positions 246, 350, 601, 605, 780, 786, 787, 800 and804 have been replaced; nevertheless, these replacements do not impairits activity in binding to the large T antigen. For example, in thepRb/p110 protein, the amino acids Thr-256, Ser-601, Ser-605, Ser-780,Ser-786, Ser-787 and Ser-800 are replaced with Ala, the amino acidThr-350 is replaced with Arg and the amino acid Ser-804 is replaced withGlu.

The DNA sequence for the p107 protein or the p130 protein is mutated inan analogous manner.

Protein p53 is inactivated in the cell either by binding to specialproteins, such as MDM2, or by oligomerization of the p53 by way of thedephosphorylated C-terminal serine 392 (Schikawa et al., Leukemia andLymphoma 11, 21 (1993) and Brown, Annals of Oncology 4, 623 (1993)).Consequently, a p53 cDNA sequence is preferably used which is truncatedC-terminally by removing the serine 392.

b) Angiogenesis inhibitors

An antitumoral substance is furthermore to be understood to be the DNAsequence for a protein which inhibits angiogenesis. Particular examplesof these proteins (the relevant DNA sequence is referred to in the citedliterature) are:

plasminogen activator inhibitor 1 (PAI-1) (Reilly et al., J. Biol. Chem.265, 9570 (1990), Bosma et al., J. Biol. Chem. 253, 9219 (1988))

PAl-2 (Steven et al., Eur. J. Biochem. 196, 431 (1991))

PAl-3 (Meijers et al., J. Biol. Chem. 266, 15028 (1991))

angiostatin (O'Reilly et al., Cell 79, 315 (1994), Bicknell et al.,Semin. Cancer Biol. 3, 399 (1992))

interferons (Dorr, Drugs 45, 177 (1993), Drugs of Today 22, 597 (1986),US 45 185 84, US 45 88 585), specifically

IFNα (Henco et al., J. Mol. Biol. 185, 227 (1985), Pestka et al., Ann.Rev. Biochem. 56, 727 (1987), Weissmann et al., Phil. Trans. R. Soc.Lond. B299, 7 (1982), Goeddel et al., Nature 290, 20 (1981))

IFNβ (Sen et al., J. Biol. Chem. 267, 5017 (1992), Mark et al., EP 192811, EP 234 599, US 45 88 585)

IFNγ (Gray et al., Nature 295, 503 (1982), Yip et al., PNAS USA 79, 1820(1982), Rinderknecht et al., J. Biol. Chem. 259, 6790 (1984))

platelet factor 4 (Mc Manus et al., J. Immunol. 123, 2835 (1979);Brindley et al., J. Clin. Invest. 72, 1218 (1983); Deuel et al., Proc.Natl. Acad. Sci. USA 78, 4584, 1981))

IL-12 (Voest et al., N. Natl. Cancer Inst. 87, 581 (1995), Wolf et al.,J. Immunol. 146, 3074 (1991), Gubler et al., PNAS USA 88, 4143 (1991),Kobayashi et al., J. Exp. Med. 170, 827 (1989), Gabler et al., PNAS 88,4143 (1991), Gately et al., J. Immunol. 147, 874 (1991), Schoenhaut etal., J. Immunol. 148, 3433 (1992), Wolf et al., J. Immunol. 146, 3074(1991))

TIMP-1 (Kolkenbrock et al., Eur. J. Biochem. 198, 775 (1991), Faucher etal., Path. Biol. 37, 199 (1989))

TIMP-2 (Kolkenbrock et al., Eur. J. Biochem. 198, 775 (1991))

TIMP-3 (Wick et al., J. Biol Chem. 269, 18953 (1994)).

leukemia inhibitory factor (LIF) (Pepper et al., J. Cell Science 108, 73(1995), Gough et al., Ciba Found. Symp. 167, 24 (1992), PNAS USA 85,5971 (1988), Stahl et al., J. Biol. Chem. 265, 8833 (1990), Rathjan etal., Cell 62, 1105 (1990))

c) Cytostatic and cytotoxic proteins

However, an antitumoral substance is also to be understood to be a DNAsequence for a protein which directly or indirectly exhibit a cytostaticeffect on tumors. These proteins include, in particular

perforin (Lin et al., Immunol. Today 16, 194 (1995))

granzyme (Smyth et al., Immunol. Today 16, 202 (1995))

IL-2 (Fietscher et al., Lymphok. Res. 6, 45 (1987), Matsui et al.,Lymphokines 12, 1 (1985), Tanaguchi et al., Nature 302, 305 (1983))

IL-4 (Lee et al., PNAS 83, 2061 (1986); Paul, Blood 77, 1859 (1991),Yokota et al., PNAS USA 83, 5894 (1986), van Leuven et al., Blood 73,1142 (1989), Arai et al., J. Immunol. 42, 274 (1989)

IL-12 (Kobayashi et al., J. Exp. Med. 170, 827 (1989), Gabler et al.,PNAS 88, 4143 (1991), Gately et al., J. Immunol. 147, 874 (1991),Schoenhaut et al., J. Immunol. 148, 3433 (1992), Wolf et al., J.Immunol. 146, 3074 (1991))

interferons, such as

IFN-α (Henco et al., J. Mol. Biol. 185, 227 (1985), Pestka et al., Ann.Rev. Biochem. 56, 727 (1987), Weissmann et al., Phil. Trans. R. Soc.Lond. B299, 7 (1982), Goeddel et al., Nature 290, 20 (1981))

IFNβ (Sen et al., J. Biol. Chem. 267, 5017 (1992), Mark et al., EP 192811, EP 234 599, US 45 88 585)

IFNγ (Gray et al., Nature 295, 503 (1982), Yip et al., PNAS USA 79, 1820(1982), Rinderknecht et al., J. Biol. Chem. 259, 6790 (1984))

TNF (Porter, TiBTech 9, 158 (1991); Sidhu et al., Pharmac. Ther. 57, 79(1993)), especially

TNFα (Beutler et al., Nature 320, 584 (1986). Kriegler et al., Cell 53,45 (1988))

TNFβ (Gray et al., Nature 312, 721 (1984), Li et al., J. Immunol. 138,4496 (1987), Aggarwal et al., J. Biol. Chem. 260, 2334 (1985))

oncostatin M (Brown et al., J. Immunol. 147, 2175 (1991); Grove et al.,J. Biol. Chem. 266, 18194 (1991); Hamilton et al., Biochem. Biophys.Res. Commun. 180, 652 (1991), Malik et al., Mol. Cell. Biol. 9, 2847(1989), Kallstad et al., J. Biol. Chem. 266, 8940 (1991))

d) Inflammation inducers

An antitumoral substance is furthermore to be understood to be the DNAsequence for a protein which, where appropriate in addition to theantitumoral effect, stimulate inflammations and thereby contributes toeliminating tumor cells. Particular examples of these proteins are:

RANTES (MCP-2) (Schall et al., J. Immunol. 141, 1018 (1988), Cell 61,361 (1990))

monocyte chemotactic and activating factor (MCAF) (Zachariae et al., J.Exp. Med. 171, 2177 (1990); Rollins et al., Blood 78, 1112 (1991);Mukaida et al., J. Immunol. 146, 1212 (1991); Sica et al., J. Immunol.144, 3034 (1990))

IL-8 (Lotz et al., J. Immunol. 148, 466 (1992); Clore et al.,Biochemistry 29, 1689 (1990), Matsushima et al., J. Exp. Med. 167, 1883(1988), Baglioni et al., J. Clin. Invest. 84, 1045 (1989), Int. J.Immunopharm. 17, 103 (1995), Carre et al., J.Clin. Invest. 88, 1802(1991))

macrophage inflammatory protein 1 (MIP-1 ∝ and MIP-1β) (Broxmeyer etal., J. Immunol. 147, 2586 (1991); Oh et al., J. Immunol. 147, 2978(1991); Graham et al., Nature 344, 442 (1990))

neutrophil activating protein 2 (NAP-2) (Walz, Rheum. Arthr., London(1991), Chang et al., J. Biol. Chem. 269, 25277 (1994))

IL-3 (Otsuka et al., J. Immunol. 140, 2288 (1988); de Vries et al., StemCells 11, 72 (1993), Yang et al., Blood 71, 958 (1988), Cell 47, 3(1986), Philips et al., Gene 84, 501 (1989))

IL-5 (Azuma et al., Nucl. Acid Res. 14, 9149 (1986); Yokota et al., PNAS84, 7388 (1987); Campbell et al., PNAS 84, 6629 (1987), Azuma et al.,Nucl. Acids Res. 14, 9149 (1986))

human leukemia inhibitory factor (LIF) (Gough et al., Ciba Found. Symp.167, 24 (1992), PNAS USA 85, 5971 (1988), Stahl et al., J. Biol. Chem.265, 8833 (1990), Rahtjan et al., Cell 62, 1105 (1990))

IL-7 (Matzuda et al., Leuk. Lymph. 5, 231 (1991), Lupton et al., J.Immunol. 144, 3592 (1990), Goodwin et al., PNAS USA 86, 302 (1989),Swatherland et al., Hum. Genet. 82, 371 (1989))

IL-11 (Paul et al., Proc. Natl. Acad. Sci. USA 87, 7512 (1990), Teramuraet al., Blood 79, 327 (1992), Kawashima et al., FEBS Lett. 283, 199(1991))

IL-13 (McKenzie et al., J. Immunol. 150, 5436 (1993), Muzio et al.,Blood 83, 1738 (1994), McKenzie et al., PNAS 90, 3735 (1993), Minty etal., Nature 362, 248 (1993))

GM-CSF (Wong et al., Science 228, 810 (1985), Gough et al., Nature 309,763 (1984), Nicola et al., J. Biol. Chem. 254, 5290 (1979))

G-CSF (Nagata et al., EMBO J. 5, 575 (1986), Nagata et al., Nature 319415 (1986), Souza et al., Science 232 61(1986))

M-CSF (Welte et al., PNAS 82, 1526 (1985), Lu et al., Arch. Biochem.Biophys. 268, 81 (1989), Kawasaki et al., Science 230, 291 (1985), Suzuet al., J. Biol. Chem. 267, 4345 (1992), Wong et al., Science 235, 1504(1987))

DNA sequences of fusion proteins which are formed between the listedcytokines, on the one hand, and the Fc moiety of human immunoglobulin,on the other hand, may also be used as active substances within themeaning of the invention. DNA sequences of this nature, and theirpreparation, have been described in EPA 0464 633 A1.

e) Enzymes for activating precursors of cytostatic agents

However, an antitumoral substance is also to be understood as being theDNA sequence of an enzyme which is able to convert precursors of anantitumoral active compound into an antitumoral active compound. Enzymesof this nature, which cleave inactive precursor substances (prodrugs)and thereby form active cytostatic agents (drugs), and the prodrugs anddrugs which are in each case pertinent, have already been reviewed byDeonarain et al. (Br. J. Cancer 70, 786 (1994), by Mullen, Pharmac.Ther. 63, 199 (1994) and by Harris et al., Gene Ther. 1, 170 (1994)).

For example, use is to be made of the DNA sequence for one of thefollowing enzymes:

herpes simplex virus thymidine kinase (Garapin et al., PNAS USA 76, 3755(1979), Vile et al., Cancer Res. 53, 3860 (1993), Wagner et al., PNASUSA 78, 1441 (1981), Moelten et al., Cancer Res. 46, 5276 (1986), J.Natl. Cancer Inst. 82, 297 (1990))

varicella zoster virus thymidine kinase (Huber et al., PNAS USA 88, 8039(1991), Snoeck, Int. J. Antimicrob. Agents 4, 211 (1994))

bacterial nitroreductase (Michael et al., FEMS Microbiol. Letters 124,195 (1994), Bryant et al., J. Biol. Chem. 266, 4126 (1991), Watanabe etal., Nucleic Acids Res. 18, 1059 (1990))

bacterial β-glucuronidase (Jefferson et al., PNAS USA 83, 8447 (1986)

plant β-glucuroniase from Secale cereale (Schulz et al., Phytochemistry26, 933 (1987))

human β-glucuronidase (Bosslet et al., Br. J. Cancer 65, 234 (1992),Oshima et al., PNAS USA 84, 685 (1987))

human carboxypeptidase (CB), e.g.

mast cell CB-A (Reynolds et al., J. Clin. Invest. 89, 273 (1992))

pancreatic CB-B (Yamamoto et al., J. Biol. Chem. 267, 2575 (1992),Catasus et al., J. Biol. Chem. 270, 6651 (1995))

bacterial carboxypeptidase (Hamilton et al., J. Bacteriol. 174, 1626(1992), Osterman et al., J. Protein Chem. 11, 561 (1992))

bacterial β-lactamase (Rodrigues et al., Cancer Res. 55, 63 (1995),Hussain et al., J. Bacteriol. 164, 223 (1985), Coque et al., Embo J. 12,631 (1993)

bacterial cytosine deaminase (Mullen et al., PNAS USA 89, 33 (1992),Austin et al., Mol. Pharmac. 43, 380 (1993), Danielson et al., Mol.Microbiol. 6, 1335 (1992)

human catalase or peroxidase (Ezurum et al., Nucl. Acids Res. 21, 1607(1993))

phosphatase, in particular

human alkaline phosphatase (Gum et al., Cancer Res. 50, 1085 (1990))

human acid prostate phosphatase (Sharieff et al., Am. J. Hum. Gen. 49,412 (1991), Song et al., Gene 129, 291 (1993), Tailor et al., Nucl.Acids Res. 18, 4928 (1990))

type 5 acid phosphatase (Gene 130, 201 (1993))

oxidase, in particular

human lysyl oxidase (Kimi et al., J. Biol. Chem. 270, 7176 (1995))

human acid D-amino oxidase (Fukui et al., J. Biol. Chem. 267, 18631(1992))

peroxidase, in particular

human gluthatione peroxidase (Chada et al., Genomics 6, 268 (1990),Ishida et al., Nucl. Acids Res. 15, 10051 (1987))

human eosinophilic peroxidase (Ten et al., J. Exp. Med. 169, 1757(1989), Sahamaki et al., J. Biol. Chem. 264, 16828 (1989))

human thyroid peroxidase (Kimura, PNAS USA 84, 5555 (1987)).

In order to facilitate secretion of the listed enzymes, the homologoussignal sequence which is in each case containing in the DNA sequence canbe replaced by a heterologous signal sequence which improvesextracellular secretion.

Thus, for example, the signal sequence of β-glucuronidase (DNA position≦27 to 93; Oshima et al., PNAS 84, 685 (1987)) can be replaced by thesignal sequence for immunoglobulin (DNA position ≦63 to ≧107; Riechmannet al., Nature 332, 323 (1988)).

In addition, DNAs are preferably to be chosen of those enzymes which, asa result of point mutations, are stored to a lesser extent in lysosomesand are secreted to an increased extent. Point mutations of this naturehave been described, for example, for β-glucuronidase (Shiplex et al.,J. Biol. Chem. 268, 12193 (1993)).

2.4. Combination of Several Antitumoral Substances

The invention furthermore relates to an active compound in which the DNAsequences of several identical antitumoral substances (A,A) or ofdifferent antitumoral substances (A,B) are combined. For the expressionof two DNA sequences, the cDNA of an internal ribosome entry site (IRES)is preferably interposed as a regulatory element. ##STR2##

Such IRESs have been described, for example, by Mountford and Smith (TIG11, 179 (1995), Kaufman et al., Nucl. Acids Res. 19, 4485 (1991), Morganet al., Nucl. Acids Res. 20, 1293 (1992, Dirks et al., Gene 128, 247(1993), Pelletier and Sonenberg, Nature 334, 320 (1988) and Sugitomo etal., BioTechn. 12, 694 (1994).

Thus, the cDNA of the IRES sequence of poliovirus (position ≦140 to ≧630of the 5' UTR; Pelletier and Sonenberg, Nature 334, 320 (1988)) can beused to link the DNA of anti-inflammatory substance A (at the 3' end)and the DNA of anti-inflammatory substance B (at the 5' terminus).

Depending on the combination (A+A, A+B1) an active compound of thisnature exhibits either an additive or a synergistic effect within themeaning of the invention.

2.5. Construction of the Vector

The novel DNA construct is made into a complete vector in a manner withwhich the skilled person is familiar. This vector can be of viral ornon-viral origin. For example, the novel DNA construct is inserted intoa viral vector (in this regard, see D. Jolly, Cancer Gene Therapy 1, 51(1994)) or else is used as a plasmid. Viral vectors or plasmids can becomplexed with colloidal dispersions, for example with liposomes(Farhood et al., Annals of the New York Academy of Sciences 716, 23(1994)), or else formulated as pharmaceuticals together with apolylysine/ligand conjugate (Curiel et al., Annals of the New YorkAcademy of Sciences 716, 36 (1994)) or other customary auxiliarysubstances.

2.6. Choice of the Ligand

Viral and non-viral vectors can be supplemented with a ligand.Substances which bind to the surface of proliferating endothelial cellsare preferred as ligands, for example in polylysine/ligand conjugates.These substances include antibodies or antibody fragments which aredirected against membrane structures of endothelial cells, as have beendescribed, for example, by Burrows et al. (Pharmac. Ther. 64, 155(1994)), Hughes et al. (Cancer Res. 49, 6214 (1989) and Maruyama et al.(PNAS-USA 87, 5744 (1990). These substances in particular includeantibodies against VEGF receptors.

The murine monoclonal antibodies are preferably to be employed inhumanized form. They are humanized in the manner described by Winter etal. (Nature 349, 293 (1991) and Hoogenbooms et al. (Rev. Tr. Transfus.Hemobiol. 36, 19 (1993)). Antibody fragments are prepared in accordancewith the state of the art, for example in the manner described by Winteret al., Nature 349, 293 (1991), Hoogenboom et al., Rev. Tr. Transfus.Hemobiol. 36, 19 (1993), Girol, Mol. Immunol. 28, 1379 (1991) or Hustonet al., Intern. Rev. Immunol. 10, 195 (1993).

The ligands furthermore include all active compounds which bind tomembrane structures or membrane receptors on endothelial cells. Forexample, they include substances which contain mannose terminally and,in addition, IL-1 or growth factors, or their fragments or constituentsequences thereof, which bind to receptors which are expressed byendothelial cells, for example PDGF, bFGF, VEGF, and TGFβ (Pusztain etal., J. Pathol. 169, 191 (1993)). In addition, they include adhesionmolecules which bind to activated and/or proliferating endothelialcells. Adhesion molecules of this nature, for example SLex, LFA-1,MAC-1, LECAM-1 or VLA4, have already been described (reviews inAugustin-Voss et al., J. Cell Biol. 119, 483 (1992), Pauli et al.,Cancer Metast. Rev. 9, 175 (1990) and Honn et al., Cancer Metast. Rev.11, 353 (1992)).

2.7 Preparation of the Active Compound (Examples)

The preparation of the novel active compound is described in more detailwith the aid of the following examples:

a) Construction of the chimeric promoter endothelin 1-CDE-CHR-lnr

The human endothelin-1 promoter (position ≦-170 to ≧-10), or a variantwhich has been truncated by removing the TATA box (position ≦-170 to≧-40), are linked, at its 3' end, to the 5' terminus of the CDE-CHR-lnrmodule (position ≦-20 to ≧+121) of the human cdc25C gene (FIG. 6). Thelinkage is effected using enzymes which are known to the skilled personand which are commercially available.

b) Construction of a plasmid which contains the central component of theactive compound

The chimeric endothelin-1 promoter module/transcription unit which hasbeen described is linked, at their 3' ends, to the 5' terminus of a DNAwhich lacuna! the complete coding region of human β-glucuronidase(position ≦27 to ≧1982; Oshima et al., PNAS USA 84, 685 (1987)). ThisDNA also contains the signal sequence (22 N-terminal amino acids) whichis required for secretion. In order to facilitate the secretion from thecell, this signal sequence is preferably to be replaced with theimmunoglobulin signal sequence (position ≦63 to ≧107; Riechmann et al.,Nature 332, 323 (1988), see FIG. 7). Transcription control units and theDNA for P-glucuronidase are cloned into pUC19/19 or Bluescript-derivedplasmid vectors, which can be employed directly, or in colloidaldispersion systems, for in-vivo administration. Alternatively, thechimeric genes can be transferred into viral vectors, or other suitablevectors, and injected.

2.8 Activity of the Active Compound

Following local administration, for example at the site of the tumor, orfollowing intracranial or subarachnoid administration, or systemic,preferably intravenous or intraarterial administration, an activecompound according to the present invention enables, by means of thecombination of tissue-specific activator sequence (UAS) and cellcycle-regulated repressor module, endothelial cells, which are mainly,if not exclusively, only proliferating endothelial cells, or neighboringcells of these proliferating endothelial cells, to secrete substanceswhich either inhibit proliferation themselves or else inducethe-formation of substances which have an antiproliferative orcytostatic effect. The inhibition of endothelial cell proliferation,and/or the liberation of cytostatic substances, result(s) in inhibitionof the growth of the tumor. The active compound is well tolerated sincethe formation of the antitumoral substance is restricted by the novelactive compound to the site of tumor growth and the angiogenesis whichis caused by the tumor.

Since the active compound promises a high degree of safety, both becauseof its cell specifity and its cell cycle specifity, it can also be usedat high doses and, if necessary, repeatedly at intervals of days orweeks, for the therapy of tumor diseases.

LEGENDS TO FIGS. 1-7:

FIG. 1: Nucleotide sequence of the cdc25C promoter region together withthe protein binding sites which have been found in vivo (genomic DMSfootprinting; • (filled circles): complete constitutive protection; ∘(open circles): partial constitutive protection; * (asterisk): cellcycle-regulated, G1-specific protection). CBS: constitutive bindingsite; CDE: cell cycle-dependent element. Regions underlaid in grayindicate the Y_(c) boxes (NF-Y binding sites). Start sites are marked byfilled squares (SEQ ID NO: 7 is shown in this Figure).

FIG. 2: Specific derepression of the cdc25C promoter in G₀ by mutatingthe cdc.

FIG. 3: Diagrammatic representation of the regulation of the cdc25Cenhancer by the CDE.

FIG. 4: G₀ /G₁ -specific repression of the SV40 enhancer by the CDE. Thenumbers on the right denote induction in G2 (in comparison to thecontrol =1)

FIG. 5: Homologies in the CDE-CHR region and the 5'-situated Yc boxes,in the cdc25C, cyclin A and cdc2 promoters (SEQ ID NOS: 1-2, 5-6, and3-4, respectively).

FIG. 6: Chimeric constructs which are composed of different moieties ofthe human endothelin-1 promoter, the 3'-fused promoter module containingthe CDE and CHR repressor elements and a DNA for human β-glucuronidase(complete coding region, position ≦239 -≧2194; Oshima et al., PNAS USA84, 685 (1987) as effector. Position designations refer to thedesignations of Wilson et al., Mol. Cell. Biol. 10, 4854 (1990) for theendothelin 1 gene and to the system for cdc25C used by Lucibello et al.,EMBO J. 15, 132 (1995), respectively.

FIGS. 7A-7B: Position designation for the signal sequence(MGWSCIILFLVATAT, SEQ ID NO: 8) of the immunoglobulin (HuVHCAMP) referto Riechmann et al., Nature 332, (1988)

Alternative: Insertion of the Ig signal peptide in order to obtainimproved extracellular secretion of the β-glucuronidase (FIG. 7B)

                  TABLE 1                                                         ______________________________________                                        Role of CDE and CHR in the cell cycle-regulated transcription of              cdc25C, cyclin A and cdc2                                                     Tab. 1                                                                                  G.sub.0  Growing  Factor                                            ______________________________________                                        wt                                                                            cdc25C      0.8        13.1     17.5                                          cyclin A    0.7        27.1     41.7                                          cdc2        1.0        41.2     41.2                                          mCDE(-13)                                                                     cdc25C      7.6        11.6     1.5                                           cyclin A    13.4       23.9     1.8                                           cdc2        11.3       33.9     3.0                                           mCHR(-6/-3)                                                                   cdc25C      14.4       21.0     1.5                                           cyclin A    15.5       28.3     1.8                                           cdc2        18.6       38.6     2.1                                           ______________________________________                                    

The results of transient transfections in HIH3T3 cells are depicted asRLUs/1000. mCDE: mutated CDE (Pos. -13:G→T); mCHR: mutated CHR (Pos. -6to -3).

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 8                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GCGCGCGGAGATTGGCTGACG21                                                       (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GGCGAGCGGGGATAGGT17                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CTGGGGTCTGATTGGCTGCTT21                                                       (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CGGGCTACCCGATTGGT17                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CTGTCGCCTTGAATGACGTCA21                                                       (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CGAGCGCTTTCATTGGT17                                                           (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 375 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       TTCGTGGGGCTGAGGGAACGAGGAAAACAGAAAGGGTGTGGAGATTGGTGAGAGGGAGAG60                CCAATGATGCGCCAGGCTCCCCGTGAGGCGGAGCTTACCCCGCAGCCTGCCTAACGCTGG120               TGGGCCAAACACTATCCTGCTCTGGCTATGGGGCGGGGCAAGTCTTACCATTTCCAGAGC180               AAGCACACGCCCCCAGGTGATCTGCGAGCCCAACGATAGGCCATGAGGCCCTGGGCGCGC240               GCGCGGAGATTGGCTGACGCAGCTTAGAGGCGAGCGGGGATAGGTTACTGGGCTGGCGGA300               AGGTTTGAATGGTCAACGCCTGCGGCTGTTGATATTCTTGCTCAGAGGCCGTAACTTTGG360               CCTTCTGCTCAGGGA375                                                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       MetGlyTrpSerCysIleIleLeuPheLeuValAlaThrAlaThr                                 151015                                                                        __________________________________________________________________________

What is claimed is:
 1. A DNA construct for the prophylaxis or therapy oftumor diseases, which comprises an activator sequence, a cellcycle-regulated promoter module, and a DNA sequence encoding anantitumor substance, wherein said promoter module comprises a cell cycledependent element, a cell cycle homology region, and an initiation site.2. A DNA construct as claimed in claim 1, wherein said promoter modulecomprises the DNA sequence of base 291 through base 340 of SEQ ID NO:7.3. A DNA construct as claimed in claim 1, wherein said cell cycledependent element has the nucleotide sequence TGGCGG.
 4. A DNA constructas claimed in claim 1, wherein said cell cycle gene homology region hasthe nucleotide sequence GTTTGAA.
 5. A DNA construct as claimed in claim1, wherein said activator sequence is regulated by transcription factorswhich are formed in endothelial cells or in cells which are in theimmediate vicinity of proliferating endothelial cells.
 6. A DNAconstruct as claimed in claim 1, wherein said activator sequence isselected from the group consisting of: the CMV promoter, the CMVenhancer and the SV40 promoter.
 7. A DNA construct as claimed in claim1, wherein said activator sequence is a promoter for a gene encoding aprotein selected from the group consisting of endothelial glucose-1transporter, endoglin, VEGF receptor 1, VEGF receptor 2, receptortyrosine kinase til-1, receptor tyrosine kinase til-2, B61 receptor, B61ligand, endothelin, endothelin B, endothelin 1, mannose 6-phosphatereceptor, IL-1∝, IL-1β, IL-1 receptor, VCAM-1, and von Willebrandfactor.
 8. A DNA construct as claimed in claim 1, wherein said activatorsequence is an oligomerized binding site for a transcription factorwhich is preferentially active in endothelial cells.
 9. A DNA constructas claimed in claim 8, wherein said binding site is the sequence5'-TTATCT-3'.
 10. A DNA construct as claimed in claim 1, wherein saidactivator sequence is selected from the group consisting of the VEGFpromoter, the VEGF enhancer, the cDNA encoding c-SRC, and the cDNAencoding v-SRC.
 11. A DNA construct as claimed in claim 1, wherein saidactivator sequence is selected from the group consisting of the promoterfor mouse mammary tumor virus and the promoter for a steroid receptorgene.
 12. A DNA construct as claimed in claim 1, wherein said antitumorsubstance is selected from the group consisting of retinoblastomaprotein p110, retinoblastoma protein p107, and retinoblastoma proteinp130.
 13. A DNA construct as claimed in claim 1, wherein said antitumorsubstance is protein p53.
 14. A DNA construct as claimed in claim 1,wherein said antitumor substance is selected from the group consistingof a cycline-dependent kinase inhibitor, protein p21, and protein p16.15. A DNA construct as claimed in claim 1, wherein said antitumorsubstance is GADD45 protein.
 16. A DNA construct as claimed in claim 1,wherein said antitumor substance is bak protein.
 17. A DNA construct asclaimed in claim 1, wherein said antitumor substance is a protein whichinhibits angiogenesis.
 18. A DNA construct as claimed in claim 1,wherein said antitumor substance is a protein which exhibits acytostatic effect.
 19. A DNA construct as claimed in claim 1, whereinsaid antitumor substance is a protein which stimulates inflammation. 20.A DNA construct as claimed in claim 1, wherein said antitumor substanceis an enzyme that cleaves a precursor of a cytostatic agent to produce acytostatic agent.
 21. A DNA construct as claimed in claim 12, whereinamino acids Thr-246, Thr-350, Ser-601, Ser-605, Ser-780, Ser-786,Ser-787, Ser-800, and Ser-804 of retinoblastoma protein p110, p107 orp130 are replaced with non-naturally occurring amino acids, and whereinsaid protein lacks phosphorylation sites, and wherein said protein iscapable of binding the large T-antigen.
 22. A DNA construct as claimedin claim 21, wherein amino acids Thr-246, Ser-601, Ser-605, Ser-780,Ser-786, Ser-787, and Ser-800 are replaced with Ala, and wherein Thr-350is replaced with Arg and wherein Ser-804 is replaced with Glu.
 23. A DNAconstruct as claimed in claim 13, wherein said protein p53 is lackingthe C-terminal Ser-392.
 24. A DNA construct as claimed in claim 1,wherein said antitumor substance is selected from the group consistingof plasminogen activator inhibitor 1, plasminogen activator inhibitor 2,plasminogen activator inhibitor 3, angiostatin, platelet factor 4,TIMP-1, TIMP-2, and TIMP-3.
 25. A DNA construct as claimed in claim 1,wherein said antitumor substance is selected from the group consistingof perforin, granzyme, IL-2, IL-4, IL-12, an interferon, IFNα, IFNβ,IFNgamma, TNFα, TNFβ, oncostatin M, RANTES, MCAF, IL-8, MIP-1α, MIP-1β,NAP-2, IL-3, IL-5, LIF, IL-11, and IL-13.
 26. A DNA construct as claimedin claim 1, wherein said antitumor substance is a fusion proteincomprising an immunoglobulin Fc fragment.
 27. A DNA construct as claimedin claim 1, wherein said antitumor substance is an enzyme selected fromthe group consisting of herpes simplex virus thymidine kinase, cytosinedeaminase, varicella zoster virus thymidine kinase, nitroreductase,β-glucuronidase, carboxypeptidase, lactamase, pyroglutamateaminopeptidase, D-aminopeptidase, oxidase, peroxidase, phosphatase,hydroxynitrile lyase, protease, esterase or glycosidase.
 28. A DNAconstruct as claimed in claim 27, wherein said β-glucuronidase isselected from the group consisting of human, plant and bacterialβ-glucuronidases.
 29. A DNA construct as claimed in claim 27, whereinsaid carboxypeptidase is a Pseudomonas carboxypeptidase.
 30. A DNAconstruct as claimed in claim 27, wherein said lactamase is a Bacilluscereus lactamase.
 31. A DNA construct as claimed in claim 27, whereinthe amino acid sequence of said enzyme has been mutated so that thelysosomal storage of said enzyme has been decreased relative to storageof the non-mutated enzyme.
 32. A DNA construct as claimed in claim 27,wherein the naturally occurring signal sequence of said enzyme isreplaced with a heterologous signal sequence, and wherein saidheterologous signal sequence increases the extracellular secretion ofsaid enzyme.
 33. A DNA construct as claimed in claim 1, wherein saidconstruct encodes more than one antitumor substance, and wherein eachDNA sequence encoding an antitumor substance is linked to another DNAsequence encoding an antitumor substance by a DNA sequence for aninternal ribosome entry site.
 34. A vector comprising a DNA construct asclaimed in claim
 1. 35. A vector as claimed in claim 34, wherein thevector is a virus.
 36. A vector as claimed in claim 35, wherein thevirus is selected from the group consisting of retrovirus, adenovirus,adeno-associated virus, herpes simplex virus and vaccinia virus.
 37. Aplasmid comprising a DNA construct as claimed in claim
 1. 38. Acolloidal dispersion system comprising a DNA construct as claimed inclaim
 1. 39. A system as claimed in claim 38, wherein the systemcomprises liposomes.
 40. A system as claimed in claim 39, wherein saidsystem comprises polylysine ligands.
 41. A vector according to claim 34,further comprising a nucleotide sequence encoding a ligand which bindsto a membrane structure of endothelial cells.
 42. A vector as claimed inclaim 41, wherein said ligand is selected from the group consisting of apolyclonal antibody, a monoclonal antibody, and an antibody fragment,and wherein the variable domains of said ligand bind to membranestructures of endothelial cells.
 43. A vector as claimed in claim 41,wherein said ligand is selected from the group consisting of a cytokine,a growth factor, a fragment of a growth factor, a fragment of acytokine, a constituent sequence of a growth factor, and a constituentsequence of a cytokine, and wherein said ligand binds to receptors onsmooth muscle cells.
 44. A vector as claimed in claim 41, wherein saidligand is selected from the group consisting of an adhesion molecule,SLeX, LFA-1, MAC-1, LECAM-1 and VLA-4.
 45. A vector as claimed in claim42, wherein said membrane structure is selected from the groupconsisting of a mannose receptor, IL-1, a growth factor, PDGF, FGF,VEGF, and TGFS.
 46. A pharmaceutical preparation comprising a DNAconstruct as claimed in claim
 1. 47. A pharmaceutical preparation asclaimed in claim 46, wherein said preparation is suitable forintravenous administration.
 48. A pharmaceutical preparation as claimedin claim 46, wherein said preparation is suitable for intraarterialadministration.
 49. A pharmaceutical preparation as claimed in claim 46,wherein said preparation is suitable for intracavity injection.
 50. Apharmaceutical preparation as claimed in claim 46, wherein saidpreparation is suitable for injection into tissue.
 51. A pharmaceuticalpreparation as claimed in claim 46, wherein said preparation is suitablefor injection into gaps in tissue.
 52. A pharmaceutical preparation asclaimed in claim 46, wherein said preparation is suitable for localadministration.